Name: assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes
Uploaded: 2013-01-07T12:00:00
Description: Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

这项工作是由美国国家卫生研究院，美国国立糖尿病，消化和肾脏疾病，2R01DK59558（GN）的资助。 UAMS翻译研究所流式细胞仪核心提供了部分资金支持由国立卫生国家研究资源中心授予UL1 RR029884在UAMS。我们感谢沛沛平博士（加州大学洛杉矶分校，洛杉矶，CA）提供的一份腺病毒携带基因编码的显性负（无效）突变体的PKC-ε和，艾伦博士Samarel（Loyola大学医学中心;梅伍德，IL），用于提供一个等分PKC-ε的组成型活性的突变体的腺病毒载体编码。我们也感谢博士。彼得·帕克和彼得·萨格登（伦敦帝国学院，伦敦，英国）组成性激活PKC-ε的cDNA编码。

1。原代培养肾小管上皮分离<ol><li>麻醉兔，切除双侧肾脏，并将其放置在培养皿中充满了消毒缓冲（DMEM/F12介质）在层流罩。 </li><li>每个肾灌注用50ml准备缓冲后跟50毫升无菌磷酸盐缓冲盐水，pH 7.4（PBS）中，PBS-氧化铁溶液（45毫升+ 5毫升）。德封装的肾脏和转移他们的准备缓冲液去铁胺。 </li><li>收获皮质每个肾。 </li><li>使用15毫升的Dounce匀浆器匀化组织。超过2无菌网筛（85微米的底部和顶部235...

<ol>
	<li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+culture+conditions+stimulate+gluconeogenesis+in+primary+cultures+of+renal+proximal+tubule+cells.">Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells.</a> Am. J. Physiol. 268, C1053-C1061 (1995).</li><li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+regulates+growth+and+metabolism+of+renal+cells:+improvements+in+cell+culture.">L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.</a> Am. J. Physiol. 271, 2072-2080 (1996).</li><li>Ping, P., Takano, H., Zhang, J., Tang, X. L., Qiu, Y., Li, R. 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Renal Physiol. 301, F197-F208 (2011).</li><li>Nowak, G., Clifton, G. L., Godwin, M. L., Bakajsova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ERK1/2+pathway+mediates+oxidant-induced+decreases+in+mitochondrial+function+in+renal+cells.">Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.</a> Am. J. Physiol. Renal Physiol. 291, 840-855 (2006).</li><li>Shaik, Z. P., Fifer, E. K., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+activation+improves+oxidative+phosphorylation+in+renal+proximal+tubular+cells+following+nephrotoxicant+injury.">Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury.</a> Am. J. Physiol. Renal Physiol. 294, F423-F432 (2008).</li><li>Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#945;+and+ERK1/2+mediate+mitochondrial+dysfunction,+decreases+in+active+Na++transport,+and+cisplatin-induced+apoptosis+in+renal+cells.">Protein kinase C-&#945; and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells.</a> J. Biol. Chem. 277, 43377-43388 (2002).</li><li>Liu, X., Godwin, M. L., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-+a+inhibits+the+repair+of+oxidative+phosphorylation+after+S-(1,2-dichlorovinyl)-L-cysteine+injury+in+renal+cells.">Protein kinase C- a inhibits the repair of oxidative phosphorylation after S-(1,2-dichlorovinyl)-L-cysteine injury in renal cells.</a> Am. J. Physiol. Renal Physiol. 287, F64-F73 (2004).</li><li>Nowak, G., Price, P. M., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+a+functional+p21WAF1/CIP1+gene+accelerates+caspase-independent+apoptosis+induced+by+cisplatin+in+renal+cells.">Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.</a> Am. J. Physiol. Renal Physiol. 285, F440-F450 (2003).</li><li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+renal+cell+apoptosis:+caspase+3-dependent+and+-independent+pathways.">Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.</a> J. Pharmacol. Exp. Ther. 302, 8-17 (2002).</li></ol>

这里介绍的方法允许个人同工酶的PKC的过度表达在肾小管上皮细胞的原代培养的。这种方法有几个优势：1。它允许在一个同质的人口调查监管机制的细胞（肾小管上皮细胞），各种损伤（缺血，缺氧，氧化应激），药物和nephrotoxicants肾脏内的主要目标。 2。 在体外模型的RPTC生长在原代培养条件的改善线粒体功能，在这个类似于肾近曲小管在体内的线粒体功能。1,2 3。线粒体?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>层流罩</td><td>热电公司</td><td> FORMA 1104 </td></tr><tr><td> 2毫升和15毫升DOUNCE组织研磨机</td><td> WHEATON </td><td> 989-24607，357544 </td></tr><tr><td> 85和235微米的尼龙网</td><td>小零件</td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50毫升无菌的离心管中</td><td> BIOLOGIX </td><td> BCT-P 50BS </td></tr><tr><td> 1.5毫升的微管</td><td>萨尔斯塔特</td><td> 72.690.001 </td></tr><tr><td> 35×10毫米的无菌培养皿</td><td>康宁</td><td> 430165 </td></tr><tr><td>儒安离心机</td><td>儒安</td><td> CR3 11175704转子：儒安儒安T40 </td></tr><tr&gt; <td>可调微离心</td><td> SIGMA </td><td>模型1 - 15 </td></tr><tr><td>生物氧气检测仪</td><td> YSI注册成立</td><td> YSI型号5300A </td></tr><tr><td>单室微型氧气系统</td><td> YSI注册成立</td><td> 5356S </td></tr><tr><td>氧探头</td><td> YSI注册成立</td><td> 5331A </td></tr><tr><td>循环浴</td><td> YSI注册成立</td><td> 5310 </td></tr><tr><td>氯化钾（KCl）和标准膜包</td><td> YSI注册成立</td><td> 5775 </td></tr><tr><td>磁力搅拌器</td><td> YSI注册成立</td><td> 5222 </td></tr><tr><td>平板式记录仪</td><td> KIPP＆Zonen公司</td><td> BD 11E </td></tr><tr><td> 48孔和96孔透明板</td><td>深圳科诗特</td><td> 3548，3679 </td></tr><tr><td>恒温ŕ </td><td> Ëppendorf </td><td> 5355 21919 </td></tr><tr><td> 2000年轨道摇床MAXQ </td><td> Thermo Scientific的</td><td> SHKA 2000 </td></tr><tr><td>光谱：FLUOR加（吸收/荧光/发光读者） </td><td> Tecan公司</td><td> F129005 </td></tr><tr><td>水套美国自动流CO 2自动孵化器</td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td>的12x75毫米聚苯乙烯文化流式细胞仪试管</td><td>费舍尔品牌</td><td> 14-961-20 </td></tr><tr><td> Axioskop 水浸物镜63X / 0,90 W </td><td>卡尔·蔡司</td><td> 114846 ACHROPLAN 44 00 67 </td></tr><tr><td> DMEM / F12 </td><td> Cellgro </td><td> 99 - 830 - PB </td></tr><tr><td> DMEM / F12火腿的</td><td>西格玛</td><td> ð2906 - 1L </td></tr><tr><td>去铁胺甲磺酸</td><td>住院RA </td><td> D110 </td></tr><tr><td> I型胶原酶</td><td>沃辛顿</td><td> 4196 </td></tr><tr><td>胰蛋白酶抑制剂</td><td>西格玛</td><td> Ţ6522 - 500MG </td></tr><tr><td>碘（JC-1）5,5&#39;，6,6&#39; - 四氯-1，1&#39;，3,3&#39; - tetraethylbenzimidazolylcarbocyanine </td><td> Invitrogen公司</td><td> T3168 </td></tr><tr><td> Mitotracker红</td><td> Invitrogen公司</td><td> M22425 </td></tr><tr><td> ATP生物发光检测试剂盒海关II </td><td>罗氏公司</td><td> 11 699 709 001 </td></tr><tr><td>膜联蛋白V - FITC解决方案</td><td>产品描述BioVision </td><td> 1001 - 200 </td></tr><tr><td>流式细胞仪</td><td> BD Biosciences公司</td><td> BD FACSCalibur </td></tr></tbody></table>

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

同功酶的蛋白激酶C（PKC）家族参与了许多生理和病理过程。我们最近的数据表明，PKC调节线粒体的功能和细胞能量状态。许多报告表明激活PKC-αPKC-ε提高在缺血性心脏的线粒体功能和介导的心脏保护作用。相反，我们已经表明，PKC-α和PKC-ε参与在nephrotoxicant诱导的线粒体功能障碍和肾脏细胞中的细胞死亡。因此，本研究的目标是开发PKC同工酶可以选择性地激活或抑制，以确定其氧化磷酸化和细胞生存的调节作用，保持积极的线粒体功能的肾细胞的体外模型。培养更好的条件，导致线粒体线粒体呼吸和活动的原代培养肾小管上皮细胞（RPTC）线粒体酶类似于那些在RPTC 体内 。由于传统的转染技术（脂质体转染，电穿孔）在原代培养中是低效的，对线粒体功能产生不利影响，PKC-ε突变cDNA分别传递到RPTC通过腺病毒载体。这种做法的结果转染培养RPTC超过90％。 在这里，我们提出了评估的作用，PKC-ε的方法：1。调节线粒体形态和ATP合成，和2与相关联的功能。 RPTC在小学文化的生存期。由过度的组成性激活PKC-ε突变激活PKC-ε。 PKC-ε抑制过度的无效突变体的PKC-ε。线粒体功能的评估是通过检查呼吸，呼吸链的完整性，活动，呼吸复合物和F 0 F 1-ATP酶，ATP生成率和ATP含量。呼吸是ssessed的毛地黄皂苷-透性RPTC状态3（最大呼吸中存在的多余的基板和ADP）和非耦合呼吸。呼吸链的完整性进行评估，通过测量所有4个配合物中分离线粒体呼吸链的活动。氧化磷酸化的能力的评价通过测定线粒体膜电位，ATP生成率，和F 0 F 1-ATP酶的活性。的RPTC的能源状况进行评估，确定细胞内ATP含量。在活细胞中的线粒体形态可视化使用Mitotracker有红580，专门蓄积在线粒体内的荧光染料，和活的单层，在荧光显微镜下检查。 RPTC可行性评估采用膜联蛋白V /碘化丙啶染色后流式细胞仪测定细胞凋亡和胀亡。 这些方法允许在个别的PKC同功酶的选择性激活/禁止，以评估它们的作用，在细胞功能中的各种生理和病理条件，可以在体外再现。

图1表明，腺病毒交付的cDNA编码的组成型活性（caPKC-ε）和非活动状态（dnPKC-ε）的突变体的PKC-ε的查询结果的显着增加的蛋白水平RPTC和线粒体中的PKC-ε。用cDNA携带caPKC-ε载体感染的细胞过表达PKC-ε的磷酸化形式（有源）而感染的细胞的cDNA编码dnPKC-ε过度表达PKC-ε是不活动的（未磷酸化的）（ 图1）。存在的活性PKC-ε降低线粒体呼吸RPTC无论通电线粒体而无效的PKC-ε并无效?...

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

活化的蛋白激酶C（PKC）的同工酶与呼吸，氧化磷酸化有关的线粒体功能和对细胞活力的影响进行说明。该方法适应腺病毒技术，选择性过度表达PKC同工酶在原代培养和各种检测，以确定线粒体功能和能量状态的细胞。

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Research

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Counting and Determining the Viability of Cultured Cells

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

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Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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Viability Assays for Cells in Culture

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

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通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的